A valine to isoleucine mutation at position 322 within variable region 3 (V3) of envelope of simian immunodeficiency virus was previously shown to compensate for an inactivating valine to glycine mutation at position 448 in constant region 4 (C4) (Morrison et al., Virology 195:167-174, 1993). Cloned DNA fragments with inactivating C4 mutations were combined with complex mixtures of mutant V3 sequences and full length genomes were transfected into COS-1 cells. By cocultivating transfected cells with CEMx174 cells, we were able to identify two additional compensatory V3-C4 combinations. Changing 334 proline to leucine compensated for an inactivating 428 asparagine to lysine mutation and changing 324 isoleucine to leucine compensated for an inactivating 448 valine to glycine mutation. The double mutants replicated efficiently in CEMx174 cells, rhesus monkey peripheral blood mononuclear cells and the continously growing rhesus monkey T cell line 221-89. Surprisingly, the 324 I L and 334 P L mutations by themselves impaired SIVmac239 wild type replication in CEMx174 cells. These results confirm the cooperation between V3 and C4 sequences and they define additional specific residues participating in this cooperation.